RESUMO
Rotavirus is a well-studied RNA virus that causes severe gastroenteritis in children. During viral entry, the outer layer of the virion is shed, creating a double-layered particle (DLP) that is competent to perform viral transcription (i.e., mRNA synthesis) and launch infection. While inactive forms of rotavirus DLPs have been structurally characterized in detail, information about the transcriptionally-active DLP remains limited. Here, we used cryo-Electron Microscopy (cryo-EM) and 3D image reconstructions to compare the structures of internal protein components in transcriptionally-active versus inactive DLPs. Our findings showed that transcriptionally-active DLPs gained internal order as mRNA synthesis unfolded, while inactive DLPs remained dynamically disordered. Regions of viral protein/RNA constituents were analyzed across two different axes of symmetry to provide a more comprehensive view of moving components. Taken together, our results bring forth a new view of active DLPs, which may enable future pharmacological strategies aimed at obliterating rotavirus transcription as a therapeutic approach.
RESUMO
The rotavirus polymerase VP1 mediates all stages of viral RNA synthesis within the confines of subviral particles and while associated with the core shell protein VP2. Transcription (positive-strand RNA [+RNA] synthesis) by VP1 occurs within double-layered particles (DLPs), while genome replication (double-stranded RNA [dsRNA] synthesis) by VP1 occurs within assembly intermediates. VP2 is critical for VP1 enzymatic activity; yet, the mechanism by which the core shell protein triggers polymerase function remains poorly understood. Structural analyses of transcriptionally competent DLPs show that VP1 is located beneath the VP2 core shell and sits slightly off-center from each of the icosahedral 5-fold axes. In this position, the polymerase is contacted by the core shell at 5 distinct surface-exposed sites, comprising VP1 residues 264 to 267, 547 to 550, 614 to 620, 968 to 980, and 1022 to 1025. Here, we sought to test the functional significance of these VP2 contact sites on VP1 with regard to polymerase activity. We engineered 19 recombinant VP1 (rVP1) proteins that contained single- or multipoint alanine mutations within each individual contact site and assayed them for the capacity to synthesize dsRNA in vitro in the presence of rVP2. Three rVP1 mutants (E265A/L267A, R614A, and D971A/S978A/I980A) exhibited diminished in vitro dsRNA synthesis. Despite their loss-of-function phenotypes, the mutants did not show major structural changes in silico, and they maintained their overall capacity to bind rVP2 in vitro via their nonmutated contact sites. These results move us toward a mechanistic understanding of rotavirus replication and identify precise VP2-binding sites on the polymerase surface that are critical for its enzymatic activation.IMPORTANCE Rotaviruses are important pathogens that cause severe gastroenteritis in the young of many animals. The viral polymerase VP1 mediates all stages of viral RNA synthesis, and it requires the core shell protein VP2 for its enzymatic activity. Yet, there are several gaps in knowledge about how VP2 engages and activates VP1. Here, we probed the functional significance of 5 distinct VP2 contact sites on VP1 that were revealed through previous structural studies. Specifically, we engineered alanine amino acid substitutions within each of the 5 VP1 regions and assayed the mutant polymerases for the capacity to synthesize RNA in the presence of VP2 in a test tube. Our results identified residues within 3 of the VP2 contact sites that are critical for robust polymerase activity. These results are important because they enhance the understanding of a key step of the rotavirus replication cycle.
Assuntos
Proteínas do Capsídeo/genética , Proteínas Mutantes/química , RNA de Cadeia Dupla/química , RNA Viral/química , RNA Polimerase Dependente de RNA/química , Rotavirus , Sequência de Aminoácidos , Sítios de Ligação , Proteínas do Capsídeo/química , Proteínas do Capsídeo/metabolismo , Mutação com Perda de Função , Modelos Moleculares , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Ligação Proteica , Conformação Proteica , RNA de Cadeia Dupla/genética , RNA de Cadeia Dupla/metabolismo , RNA Viral/genética , RNA Viral/metabolismo , RNA Polimerase Dependente de RNA/genética , RNA Polimerase Dependente de RNA/metabolismo , Rotavirus/genética , Rotavirus/metabolismoRESUMO
The fight against human disease requires a multidisciplinary scientific approach. Applying tools from seemingly unrelated areas, such as materials science and molecular biology, researchers can overcome long-standing challenges to improve knowledge of molecular pathologies. Here, custom-designed substrates composed of silicon nitride (SiN) are used to study the 3D attributes of tumor suppressor proteins that function in DNA repair events. New on-chip preparation strategies enable the isolation of native protein complexes from human cancer cells. Combined techniques of cryo-electron microscopy (EM) and molecular modeling reveal a new modified form of the p53 tumor suppressor present in aggressive glioblastoma multiforme cancer cells. Taken together, the findings provide a radical new design for cryo-EM substrates to evaluate the structures of disease-related macromolecules.
Assuntos
Microscopia Crioeletrônica/métodos , Linhagem Celular Tumoral , Humanos , Imageamento Tridimensional , Substâncias Macromoleculares/química , Compostos de Silício/químicaRESUMO
Group A rotaviruses (RVAs) are classified according to a nucleotide sequence-based system that assigns a genotype to each of the 11 double-stranded RNA (dsRNA) genome segments. For the segment encoding the VP1 polymerase, 22 genotypes (R1 to R22) are defined with an 83% nucleotide identity cutoff value. For the segment encoding the VP2 core shell protein, which is a functional VP1-binding partner, 20 genotypes (C1 to C20) are defined with an 84% nucleotide identity cutoff value. However, the extent to which the VP1 and VP2 proteins encoded by these genotypes differ in their sequences or interactions has not been described. Here, we sought to (i) delineate the relationships and sites of variation for VP1 and VP2 proteins belonging to the known RVA genotypes and (ii) correlate intergenotypic sequence diversity with functional VP1-VP2 interaction(s) during dsRNA synthesis. Using bioinformatic approaches, we revealed which VP1 and VP2 genotypes encode divergent proteins and identified the positional locations of amino acid changes in the context of known structural domains/subdomains. We then employed an in vitro dsRNA synthesis assay to test whether genotype R1, R2, R4, and R7 VP1 polymerases could be enzymatically activated by genotype C1, C2, C4, C5, and C7 VP2 core shell proteins. Genotype combinations that were incompatible informed the rational design and in vitro testing of chimeric mutant VP1 and VP2 proteins. The results of this study connect VP1 and VP2 nucleotide-level diversity to protein-level diversity for the first time, and they provide new insights into regions/residues critical for VP1-VP2 interaction(s) during viral genome replication.IMPORTANCE Group A rotaviruses (RVAs) are widespread in nature, infecting numerous mammalian and avian hosts and causing severe gastroenteritis in human children. RVAs are classified using a system that assigns a genotype to each viral gene according to its nucleotide sequence. To date, 22 genotypes have been described for the gene encoding the viral polymerase (VP1), and 20 genotypes have been described for the gene encoding the core shell protein (VP2). Here, we analyzed if/how the VP1 and VP2 proteins encoded by the known RVA genotypes differ from each other in their sequences. We also used a biochemical approach to test whether the intergenotypic sequence differences influenced how VP1 and VP2 functionally engage each other to mediate RNA synthesis in a test tube. This work is important because it increases our understanding of RVA protein-level diversity and raises new ideas about the VP1-VP2 binding interface(s) that is important for viral replication.
Assuntos
Proteínas do Capsídeo/genética , Biologia Computacional/métodos , Rotavirus/classificação , Proteínas do Core Viral/genética , Sequência de Aminoácidos , Proteínas do Capsídeo/química , Evolução Molecular , Variação Genética , Genótipo , Modelos Moleculares , Filogenia , Rotavirus/genética , Rotavirus/metabolismo , Proteínas do Core Viral/químicaRESUMO
Rotaviruses (RVs) can evolve through the process of reassortment, whereby the 11 double-stranded RNA genome segments are exchanged among strains during co-infection. However, reassortment is limited in cases where the genes or encoded proteins of co-infecting strains are functionally incompatible. In this study, we employed a helper virus-based reverse genetics system to identify NSP2 gene regions that correlate with restricted reassortment into simian RV strain SA11. We show that SA11 reassortants with NSP2 genes from human RV strains Wa or DS-1 were efficiently rescued and exhibit no detectable replication defects. However, we could not rescue an SA11 reassortant with a human RV strain AU-1 NSP2 gene, which differs from that of SA11 by 186 nucleotides (36 amino acids). To map restriction determinants, we engineered viruses to contain chimeric NSP2 genes in which specific regions of AU-1 sequence were substituted with SA11 sequence. We show that a region spanning AU-1 NSP2 gene nucleotides 784-820 is critical for the observed restriction; yet additional determinants reside in other gene regions. In silico and in vitro analyses were used to predict how the 784-820 region may impact NSP2 gene/protein function, thereby informing an understanding of the reassortment restriction mechanism.
Assuntos
Proteínas de Ligação a RNA/genética , Vírus Reordenados/genética , Recombinação Genética , Rotavirus/genética , Proteínas não Estruturais Virais/genética , Animais , Linhagem Celular , Análise Mutacional de DNA , Genoma Viral , Haplorrinos , Humanos , Vírus Reordenados/crescimento & desenvolvimento , Genética Reversa , Rotavirus/crescimento & desenvolvimento , Replicação ViralRESUMO
Temperature-sensitive (ts) mutants of simian rotavirus (RV) strain SA11 have been previously created to investigate the functions of viral proteins during replication. One mutant, SA11-tsC, has a mutation that maps to the gene encoding the VP1 polymerase and shows diminished growth and RNA synthesis at 39°C compared to that at 31°C. In the present study, we sequenced all 11 genes of SA11-tsC, confirming the presence of an L138P mutation in the VP1 N-terminal domain and identifying 52 additional mutations in four other viral proteins (VP4, VP7, NSP1, and NSP2). To investigate whether the L138P mutation induces a ts phenotype in VP1 outside the SA11-tsC genetic context, we employed ectopic expression systems. Specifically, we tested whether the L138P mutation affects the ability of VP1 to localize to viroplasms, which are the sites of RV RNA synthesis, by expressing the mutant form as a green fluorescent protein (GFP) fusion protein (VP1L138P-GFP) (i) in wild-type SA11-infected cells or (ii) in uninfected cells along with viroplasm-forming proteins NSP2 and NSP5. We found that VP1L138P-GFP localized to viroplasms and interacted with NSP2 and/or NSP5 at 31°C but not at 39°C. Next, we tested the enzymatic activity of a recombinant mutant polymerase (rVP1L138P) in vitro and found that it synthesized less RNA at 39°C than at 31°C, as well as less RNA than the control at all temperatures. Together, these results provide a mechanistic basis for the ts phenotype of SA11-tsC and raise important questions about the role of leucine 138 in supporting key protein interactions and the catalytic function of the VP1 polymerase.IMPORTANCE RVs cause diarrhea in the young of many animal species, including humans. Despite their medical and economic importance, gaps in knowledge exist about how these viruses replicate inside host cells. Previously, a mutant simian RV (SA11-tsC) that replicates worse at higher temperatures was identified. This virus has an amino acid mutation in VP1, which is the enzyme responsible for copying the viral RNA genome. The mutation is located in a poorly understood region of the polymerase called the N-terminal domain. In this study, we determined that the mutation reduces the ability of VP1 to properly localize within infected cells at high temperatures, as well as reduced the ability of the enzyme to copy viral RNA in a test tube. The results of this study explain the temperature sensitivity of SA11-tsC and shed new light on functional protein-protein interaction sites of VP1.
Assuntos
Rotavirus/enzimologia , Proteínas do Core Viral/genética , Sequência de Aminoácidos , Animais , Células COS , Chlorocebus aethiops , Estabilidade Enzimática , Simulação de Dinâmica Molecular , Mutação de Sentido Incorreto , Ligação Proteica , Domínios Proteicos , Transporte Proteico , Temperatura , Proteínas do Core Viral/química , Proteínas do Core Viral/metabolismoRESUMO
Shared VH1-46 gene usage has been described in B cells reacting to desmoglein 3 (Dsg3) in the autoimmune disease pemphigus vulgaris (PV), as well as B cells responding to rotavirus capsid protein VP6. In both diseases, VH1-46 B cells bearing few to no somatic mutations can recognize the disease Ag. This intriguing connection between an autoimmune response to self-antigen and an immune response to foreign Ag prompted us to investigate whether VH1-46 B cells may be predisposed to Dsg3-VP6 cross-reactivity. Focused testing of VH1-46 mAbs previously isolated from PV and rotavirus-exposed individuals indicates that cross-reactivity is rare, found in only one of seven VH1-46 IgG clonotypes. High-throughput screening of IgG B cell repertoires from two PV patients identified no additional cross-reactive clonotypes. Screening of IgM B cell repertoires from one non-PV and three PV patients identified specific cross-reactive Abs in one PV patient, but notably all six cross-reactive clonotypes used VH1-46. Site-directed mutagenesis studies indicate that amino acid residues predisposing VH1-46 Abs to Dsg3 reactivity reside in CDR2. However, somatic mutations only rarely promote Dsg3-VP6 cross-reactivity; most mutations abolish VP6 and/or Dsg3 reactivity. Nevertheless, functional testing identified two cross-reactive VH1-46 Abs that both disrupt keratinocyte adhesion and inhibit rotavirus replication, indicating the potential for VH1-46 Abs to have both pathologic autoimmune and protective immune functions. Taken together, these studies suggest that certain VH1-46 B cell populations may be predisposed to Dsg3-VP6 cross-reactivity, but multiple mechanisms prevent the onset of autoimmunity after rotavirus exposure.
Assuntos
Antígenos Virais/imunologia , Autoantígenos/imunologia , Proteínas do Capsídeo/imunologia , Desmogleína 3/imunologia , Fosfatases de Especificidade Dupla/imunologia , Reações Cruzadas , Ensaio de Imunoadsorção Enzimática , Ensaios de Triagem em Larga Escala , Humanos , Microscopia de Fluorescência , Pênfigo/imunologia , Reação em Cadeia da Polimerase , Infecções por Rotavirus/imunologiaRESUMO
Segmented RNA viruses are widespread in nature and include important human, animal and plant pathogens, such as influenza viruses and rotaviruses. Although the origin of RNA virus genome segmentation remains elusive, a major consequence of this genome structure is the capacity for reassortment to occur during co-infection, whereby segments are exchanged among different viral strains. Therefore, reassortment can create viral progeny that contain genes that are derived from more than one parent, potentially conferring important fitness advantages or disadvantages to the progeny virus. However, for segmented RNA viruses that package their multiple genome segments into a single virion particle, reassortment also requires genetic compatibility between parental strains, which occurs in the form of conserved packaging signals, and the maintenance of RNA and protein interactions. In this Review, we discuss recent studies that examined the mechanisms and outcomes of reassortment for three well-studied viral families - Cystoviridae, Orthomyxoviridae and Reoviridae - and discuss how these findings provide new perspectives on the replication and evolution of segmented RNA viruses.
Assuntos
Evolução Molecular , Genoma Viral , Vírus de RNA/genética , RNA Viral/química , Vírus Reordenados/genética , Recombinação Genética , Animais , Cystoviridae/genética , Cystoviridae/fisiologia , Humanos , Vírus da Influenza A/genética , Vírus da Influenza A/fisiologia , Orthomyxoviridae/genética , Orthomyxoviridae/fisiologia , Vírus de RNA/fisiologia , RNA Viral/genética , RNA Viral/metabolismo , Vírus Reordenados/fisiologia , Reoviridae/genética , Reoviridae/fisiologia , Replicação ViralRESUMO
Group A rotaviruses (RVAs) are 11-segmented, double-stranded RNA viruses and important causes of gastroenteritis in the young of many animal species. Previous studies have suggested that human Wa-like RVAs share a close evolutionary relationship with porcine RVAs. Specifically, the VP1-VP3 and NSP2-5/6 genes of these viruses are usually classified as genotype 1 with >81% nucleotide sequence identity. Yet, it remains unknown whether the genotype 1 genes and proteins of human Wa-like strains are distinguishable from those of porcine strains. To investigate this, we performed comprehensive bioinformatic analyses using all known genotype 1 gene sequences. The RVAs analyzed represent wildtype strains isolated from humans or pigs at various geographical locations during the years of 2004-2013, including 11 newly-sequenced porcine RVAs from Brazil. We also analyzed archival strains that were isolated during the years of 1977-1992 as well as atypical strains involved in inter-species transmission between humans and pigs. We found that, in general, the genotype 1 genes of typical modern human Wa-like RVAs clustered together in phylogenetic trees and were separate from those of typical modern porcine RVAs. The only exception was for the NSP5/6 gene, which showed no host-specific phylogenetic clustering. Using amino acid sequence alignments, we identified 34 positions that differentiated the VP1-VP3, NSP2, and NSP3 genotype 1 proteins of typical modern human Wa-like RVAs versus typical modern porcine RVAs and documented how these positions vary in the archival/unusual isolates. No host-specific amino acid positions were identified for NSP4, NSP5, or NSP6. Altogether, the results of this study support the notion that human Wa-like RVAs and porcine RVAs are evolutionarily related, but indicate that some of their genotype 1 genes and proteins have diverged over time possibly as a reflection of sequestered replication and protein co-adaptation in their respective hosts.
Assuntos
Rotavirus/classificação , Análise de Sequência de RNA/métodos , Proteínas Virais/genética , Animais , Biologia Computacional/métodos , Evolução Molecular , Genótipo , Humanos , Filogenia , Rotavirus/genética , SuínosRESUMO
Rotaviruses (RVs) replicate their segmented, double-stranded RNA genomes in tandem with early virion assembly. In this study, we sought to gain insight into the ultrastructure of RV assembly-replication intermediates (RIs) using transmission electron microscopy (EM). Specifically, we examined a replicase-competent, subcellular fraction that contains all known RV RIs. Three never-before-seen complexes were visualized in this fraction. Using in vitro reconstitution, we showed that ~15-nm doughnut-shaped proteins in strings were nonstructural protein 2 (NSP2) bound to viral RNA transcripts. Moreover, using immunoaffinity-capture EM, we revealed that ~20-nm pebble-shaped complexes contain the viral RNA polymerase (VP1) and RNA capping enzyme (VP3). Finally, using a gel purification method, we demonstrated that ~30-70-nm electron-dense, particle-shaped complexes represent replicase-competent core RIs, containing VP1, VP3, and NSP2 as well as capsid proteins VP2 and VP6. The results of this study raise new questions about the interactions among viral proteins and RNA during the concerted assembly-replicase process.
Assuntos
Rotavirus/fisiologia , Rotavirus/ultraestrutura , Montagem de Vírus , Replicação Viral , Animais , Linhagem Celular , Cromatografia em Gel , Haplorrinos , Substâncias Macromoleculares/ultraestrutura , Microscopia Eletrônica de Transmissão , Microscopia Imunoeletrônica , Ligação Proteica , RNA Viral/metabolismo , Proteínas Virais/metabolismoRESUMO
The molecular mechanisms by which RNA viruses coordinate their transcriptional activities are not fully understood. For rotavirus, an important pediatric gastroenteric pathogen, transcription occurs within a double-layered particle that encloses the viral genome. To date, there remains very little structural information available for actively-transcribing rotavirus double-layered particles, which could provide new insights for antiviral development. To improve our vision of these viral assemblies, we developed a new combinatorial strategy that utilizes currently available high-resolution image processing tools. First, we employed a 3D classification routine that allowed us to sort transcriptionally-active rotavirus assemblies on the basis of their internal density. Next, we implemented an additional 3D refinement procedure using the most active class of DLPs. For comparison, the refined structures were computed in parallel by (1) enforcing icosahedral symmetry, and by (2) using no symmetry operators. Comparing the resulting structures, we were able to visualize the continuum that exists between viral capsid proteins and the viral RNA for the first time.
RESUMO
UNLABELLED: Rotaviruses (RVs) are 11-segmented, double-stranded RNA viruses that cause severe gastroenteritis in children. In addition to an error-prone genome replication mechanism, RVs can increase their genetic diversity by reassorting genes during host coinfection. Such exchanges allow RVs to acquire advantageous genes and adapt in the face of selective pressures. However, reassortment may also impose fitness costs if it unlinks genes/proteins that have accumulated compensatory, coadaptive mutations and that operate best when kept together. To better understand human RV evolutionary dynamics, we analyzed the genome sequences of 135 strains (genotype G1/G3/G4-P[8]-I1-C1-R1-A1-N1-T1-E1-H1) that were collected at a single location in Washington, DC, during the years 1974 to 1991. Intragenotypic phylogenetic trees were constructed for each viral gene using the nucleotide sequences, thereby defining novel allele level gene constellations (GCs) and illuminating putative reassortment events. The results showed that RVs with distinct GCs cocirculated during the vast majority of the collection years and that some of these GCs persisted in the community unchanged by reassortment. To investigate the influence of protein coadaptation on GC maintenance, we performed a mutual information-based analysis of the concatenated amino acid sequences and identified an extensive covariance network. Unexpectedly, amino acid covariation was highest between VP4 and VP2, which are structural components of the RV virion that are not thought to directly interact. These results suggest that GCs may be influenced by the selective constraints placed on functionally coadapted, albeit noninteracting, viral proteins. This work raises important questions about mutation-reassortment interplay and its impact on human RV evolution. IMPORTANCE: Rotaviruses are devastating human pathogens that cause severe diarrhea and kill >450,000 children each year. The virus can evolve by accumulating mutations and by acquiring new genes from other strains via a process called reassortment. However, little is known about the relationship between mutation accumulation and gene reassortment for rotaviruses and how it impacts viral evolution. In this study, we analyzed the genome sequences of human strains found in clinical fecal specimens that were collected at a single hospital over an 18-year time span. We found that many rotaviruses did not reassort their genes but instead maintained them as specific sets (i.e., constellations). By analyzing the encoded proteins, we discovered concurrent amino acid changes among them, which suggests that they are functionally coadapted to operate best when kept together. This study increases our understanding of how rotaviruses evolve over time in the human population.
Assuntos
Evolução Molecular , Rotavirus/genética , Rotavirus/isolamento & purificação , Proteínas Virais/genética , Adaptação Biológica , Pré-Escolar , Análise por Conglomerados , District of Columbia , Genoma Viral , Humanos , Lactente , Dados de Sequência Molecular , Filogenia , Rotavirus/classificação , Análise de Sequência de DNARESUMO
UNLABELLED: Rotaviruses (RVs) are leading causes of severe diarrhea and vomiting in infants and young children. RVs with G10P[11] genotype specificity have been associated with symptomatic and asymptomatic neonatal infections in Vellore, India. To identify possible viral genetic determinants responsible for differences in symptomology, the genome sequences of G10P[11] RVs in stool samples of 19 neonates with symptomatic infections and 20 neonates with asymptomatic infections were determined by Sanger and next-generation sequencing. The data showed that all 39 viruses had identical genotype constellations (G10-P[11]-I2-R2-C2-M2-A1-N1-T1-E2-H3), the same as those of the previously characterized symptomatic N155 Vellore isolate. The data also showed that the RNA and deduced protein sequences of all the Vellore G10P[11] viruses were nearly identical; no nucleotide or amino acid differences were found that correlated with symptomatic versus asymptomatic infection. Next-generation sequencing data revealed that some stool samples, both from neonates with symptomatic infections and from neonates with asymptomatic infections, also contained one or more positive-strand RNA viruses (Aichi virus, astrovirus, or salivirus/klassevirus) suspected of being potential causes of pediatric gastroenteritis. However, none of the positive-strand RNA viruses could be causally associated with the development of symptoms. These results indicate that the diversity of clinical symptoms in Vellore neonates does not result from genetic differences among G10P[11] RVs; instead, other undefined factors appear to influence whether neonates develop gastrointestinal disease symptoms. IMPORTANCE: Rotavirus (RV) strains have been identified that preferentially replicate in neonates, in some cases, without causing gastrointestinal disease. Surveillance studies have established that G10P[11] RVs are a major cause of neonatal infection in Vellore, India, with half of infected neonates exhibiting symptoms. We used Sanger and next-generation sequencing technologies to contrast G10P[11] RVs recovered from symptomatic and asymptomatic neonates. Remarkably, the data showed that the RNA genomes of the viruses were virtually indistinguishable and lacked any differences that could explain the diversity of clinical outcomes among infected Vellore neonates. The sequencing results also indicated that some symptomatic and some asymptomatic Vellore neonates were infected with other enteric viruses (Aichi virus, astrovirus, salvirus/klassevirus); however, none could be correlated with the presence of symptoms in neonates. Together, our findings suggest that other poorly defined factors, not connected to the genetic makeup of the Vellore G10P[11] viruses, influence whether neonates develop gastrointestinal disease symptoms.
Assuntos
Diarreia/virologia , Infecções por Rotavirus/virologia , Rotavirus/genética , Fezes/virologia , Gastroenterite/virologia , Genótipo , Humanos , Índia , Recém-Nascido , Kobuvirus/genéticaRESUMO
UNLABELLED: Group A rotaviruses (RVs) remain a leading cause of childhood gastroenteritis worldwide. Although the G/P types of locally circulating RVs can vary from year to year and differ depending upon geographical location, those with G1P[8], G2P[4], G3P[8], G4P[8], G9P[8], and G12P[8] specificities typically dominate. Little is known about the evolution and diversity of G2P[4] RVs and the possible role that widespread vaccine use has had on their increased frequency of detection. To address these issues, we analyzed the 12 G2P[4] RV isolates associated with a rise in RV gastroenteritis cases at Vanderbilt University Medical Center (VUMC) during the 2010-2011 winter season. Full-genome sequencing revealed that the isolates had genotype 2 constellations typical of DS-1-like viruses (G2P[4]-I2-R2-C2-M2-A2-N2-T2-E2-H2). Phylogenetic analyses showed that the genome segments of the isolates were comprised of two or three different subgenotype alleles; this enabled recognition of three distinct clades of G2P[4] viruses that caused disease at VUMC in the 2010-2011 season. Although the three clades cocirculated in the same community, there was no evidence of interclade reassortment. Bayesian analysis of 328 VP7 genes of G2 viruses isolated in the last 39 years indicate that existing G2 VP7 gene lineages continue to evolve and that novel lineages, as represented by the VUMC isolates, are constantly being formed. Moreover, G2 lineages are characteristically shaped by lineage turnover events that introduce new globally dominant strains every 7 years, on average. The ongoing evolution of G2 VP7 lineages may give rise to antigenic changes that undermine vaccine effectiveness in the long term. IMPORTANCE: Little is known about the diversity of cocirculating G2 rotaviruses and how their evolution may undermine the effectiveness of rotavirus vaccines. To expand our understanding of the potential genetic range exhibited by rotaviruses circulating in postvaccine communities, we analyzed part of a collection of rotaviruses recovered from pediatric patients in the United States from 2010 to 2011. Examining the genetic makeup of these viruses revealed they represented three segregated groups that did not exchange genetic material. The distinction between these three groups may be explained by three separate introductions. By comparing a specific gene, namely, VP7, of the recent rotavirus isolates to those from a collection recovered from U.S. children between 1974 and 1991 and other globally circulating rotaviruses, we were able to reconstruct the timing of events that shaped their ancestry. This analysis indicates that G2 rotaviruses are continuously evolving, accumulating changes in their genetic material as they infect new patients.
Assuntos
Infecções por Rotavirus/epidemiologia , Infecções por Rotavirus/virologia , Rotavirus/classificação , Rotavirus/genética , Centros Médicos Acadêmicos , Pré-Escolar , Análise por Conglomerados , Evolução Molecular , Gastroenterite/epidemiologia , Gastroenterite/virologia , Genoma Viral , Genótipo , Humanos , Lactente , Recém-Nascido , Epidemiologia Molecular , Dados de Sequência Molecular , Filogenia , Rotavirus/isolamento & purificação , Análise de Sequência de DNA , Tennessee/epidemiologiaRESUMO
Understanding the fundamental properties of macromolecules has enhanced the development of emerging technologies used to improve biomedical research. Currently, there is a critical need for innovative platforms that can illuminate the function of biomedical reagents in a native environment. To address this need, we have developed an in situ approach to visualize the dynamic behavior of biomedically relevant macromolecules at the nanoscale. Newly designed silicon nitride devices containing integrated "microwells" were used to enclose active macromolecular specimens in liquid for transmission electron microscopy imaging purposes.We were able to successfully examine novel magnetic resonance imaging contrast reagents, micelle suspensions, liposome carrier vehicles, and transcribing viral assemblies. With each specimen tested, the integrated microwells adequately maintained macromolecules in discrete local environments while enabling thin liquid layers to be produced.
Assuntos
Substâncias Macromoleculares/ultraestrutura , Microscopia Eletrônica de Transmissão/métodos , Manejo de Espécimes/métodos , Meios de Contraste/análise , Lipossomos/ultraestrutura , Micelas , Vírus/ultraestruturaRESUMO
Group A rotaviruses (RVs) are eleven-segmented, double-stranded RNA viruses and important causes of severe diarrhea in children. A full-genome classification system is readily used to describe the genetic makeup of individual RV strains. In this system, each viral gene is assigned a specific genotype based upon its nucleotide sequence and established percent identity cut-off values. However, a faster and more cost-effective approach to determine RV gene genotypes is to utilize specific oligonucleotide primer sets in RT-PCR/PCR. Such primer sets and PCR-based genotyping methods have already been developed for the VP7-, VP6-, VP4- and NSP4-coding gene segments. In this study, primers were developed for the remaining seven RV gene segments, which encode proteins VP1, VP2, VP3, NSP1, NSP2, NSP3, and NSP5/6. Specifically, primers were designed to distinguish the two most common human RV genotypes (1 vs. 2) for these genes and were validated on several cell culture-adapted human and animal RV strains, as well as on human RVs from clinical fecal specimens. As such, primer sets now exist for all eleven genes of common human RVs, allowing for the identification of reassortant strains with mixed constellations of both genotype 1 and 2 genes using a rapid and economical RT-PCR/PCR method.
Assuntos
Reação em Cadeia da Polimerase/métodos , Infecções por Rotavirus/virologia , Rotavirus/classificação , Rotavirus/genética , Virologia/métodos , Primers do DNA/genética , Gastroenterite/diagnóstico , Gastroenterite/virologia , Genótipo , Humanos , Infecções por Rotavirus/diagnóstico , Sensibilidade e EspecificidadeRESUMO
RNA viruses are ubiquitous in nature, infecting every known organism on the planet. These viruses can also be notorious human pathogens with significant medical and economic burdens. Central to the lifecycle of an RNA virus is the synthesis of new RNA molecules, a process that is mediated by specialized virally encoded enzymes called RNA-dependent RNA polymerases (RdRps). RdRps directly catalyze phosphodiester bond formation between nucleoside triphosphates in an RNA-templated manner. These enzymes are strikingly conserved in their structural and functional features, even among diverse RNA viruses belonging to different families. During host cell infection, the activities of viral RdRps are often regulated by viral cofactor proteins. Cofactors can modulate the type and timing of RNA synthesis by directly engaging the RdRp and/or by indirectly affecting its capacity to recognize template RNA. High-resolution structures of RdRps as apoenzymes, bound to RNA templates, in the midst of catalysis, and/or interacting with regulatory cofactor proteins, have dramatically increased our understanding of viral RNA synthetic mechanisms. Combined with elegant biochemical studies, such structures are providing a scientific platform for the rational design of antiviral agents aimed at preventing and treating RNA virus-induced diseases.
Assuntos
Vírus de RNA/enzimologia , RNA Viral/biossíntese , RNA Polimerase Dependente de RNA/metabolismo , Animais , Coenzimas/metabolismo , Humanos , Modelos Biológicos , Modelos Moleculares , Plantas/virologia , Conformação Proteica , RNA Polimerase Dependente de RNA/químicaRESUMO
The triple-layered rotavirus virion encases an 11-segmented, dsRNA genome and 11-12 copies of the viral polymerase (VP1). VP1 transcribes and replicates the genome while tethered beneath the VP2 core shell. Genome replication (i.e. minus-strand RNA synthesis) by VP1 occurs in association with core assembly. During this process, VP2 directly engages VP1, thereby (i) packaging the polymerase into a nascent core and (ii) triggering the enzyme to initiate minus-strand RNA synthesis on bound plus-strand RNA templates. Recent work has shed light on VP2 regions important for VP1 enzymic activity. In the current study, we sought to investigate VP2 subdomains involved in the encapsidation of VP1 into recombinant virus-like particles (VLPs), which are formed of VP2 and the middle layer virion protein (VP6). We showed that strain SA11 VLPs efficiently encapsidated SA11 VP1, but not the genetically divergent Bristol VP1. VLPs made with an SA11 VP2 mutant lacking residues 1-10 of the amino-terminal domain (NTD) were still able to encapsidate VP1; however, removal of the entire NTD (residues 1-102) completely abolished polymerase packaging. We also showed that a chimeric VP2 protein containing the NTD and dimer-forming subdomain of strain Bristol VP2 can efficiently encapsidate SA11 VP1. These results suggest that the VP2 NTD and dimer-forming subdomain play important, albeit non-specific, roles in both VP1 packaging and activation. When combined with previous work, the results of this study support the notion that the same VP2 regions that engage VP1 during activation are also involved in packaging the enzyme into the core.
Assuntos
Proteínas do Capsídeo/metabolismo , Rotavirus/fisiologia , Proteínas do Core Viral/metabolismo , Montagem de Vírus , Proteínas do Capsídeo/genética , Análise Mutacional de DNA , Humanos , Mutação , Domínios e Motivos de Interação entre Proteínas , Deleção de Sequência , Proteínas do Core Viral/genéticaRESUMO
Researchers regularly use Transmission Electron Microscopes (TEMs) to examine biological entities and to assess new materials. Here, we describe an additional application for these instruments- viewing viral assemblies in a liquid environment. This exciting and novel method of visualizing biological structures utilizes a recently developed microfluidic-based specimen holder. Our video article demonstrates how to assemble and use a microfluidic holder to image liquid specimens within a TEM. In particular, we use simian rotavirus double-layered particles (DLPs) as our model system. We also describe steps to coat the surface of the liquid chamber with affinity biofilms that tether DLPs to the viewing window. This permits us to image assemblies in a manner that is suitable for 3D structure determination. Thus, we present a first glimpse of subviral particles in a native liquid environment.